scholarly journals Prophylaxis and Therapy of Inhalational Anthrax by a Novel Monoclonal Antibody to Protective Antigen That Mimics Vaccine-Induced Immunity

2006 ◽  
Vol 74 (10) ◽  
pp. 5840-5847 ◽  
Author(s):  
Laura Vitale ◽  
Diann Blanset ◽  
Israel Lowy ◽  
Thomas O'Neill ◽  
Joel Goldstein ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Protective Antigen ◽  
Fc Receptor ◽  
Anthrax Toxin ◽  
Functional Assay ◽  
Human Mabs ◽  
Anthrax Spores ◽  
Anthrax Vaccines

ABSTRACT The neutralizing antibody response to the protective antigen (PA) component of anthrax toxin elicited by approved anthrax vaccines is an accepted correlate for vaccine-mediated protection against anthrax. We reasoned that a human anti-PA monoclonal antibody (MAb) selected on the basis of superior toxin neutralization activity might provide potent protection against anthrax. The fully human MAb (also referred to as MDX-1303 or Valortim) was chosen from a large panel of anti-PA human MAbs generated using transgenic mice immunized with recombinant PA solely on the basis of in vitro anthrax toxin neutralization. This MAb was effective in prophylactic and postsymptomatic treatment of rabbits exposed to aerosolized anthrax spores, and a single intramuscular injection of 1 mg/kg of body weight fully protected cynomolgus monkeys challenged with aerosolized anthrax spores. Importantly, MAb 1303 defines a novel neutralizing epitope that requires Fc receptor engagement for maximal activity. F(ab′)2 fragments of MAb 1303, which retain equivalent affinity for PA, are 10- to 100-fold less potent in neutralizing anthrax toxin in vitro. Addition of Fc receptor-blocking antibodies also greatly reduced the activity of MAb 1303. Moreover, we found that the neutralizing activity of mouse, rabbit, and human antisera elicited by PA vaccines was effectively abrogated by blocking Fc receptors. Selection of an anti-PA MAb by using a functional assay that is a surrogate for protection has resulted in the identification of a fully human MAb with potent activity in vivo and uncovered a previously unrecognized mechanism of antibody-mediated toxin neutralization that is important for currently used anthrax vaccines.

scholarly journals Detection of Anthrax Toxin in the Serum of Animals Infected with Bacillus anthracis by Using Engineered Immunoassays

2006 ◽  
Vol 13 (6) ◽  
pp. 671-677 ◽  
Author(s):  
Robert Mabry ◽  
Kathleen Brasky ◽  
Robert Geiger ◽  
Ricardo Carrion ◽  
Gene B. Hubbard ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Systemic Circulation ◽  
Rapid Identification ◽  
Anthrax Toxin ◽  
Soluble Form ◽  
Before And After

ABSTRACT Several strategies that target anthrax toxin are being developed as therapies for infection by Bacillus anthracis. Although the action of the tripartite anthrax toxin has been extensively studied in vitro, relatively little is known about the presence of toxins during an infection in vivo. We developed a series of sensitive sandwich enzyme-linked immunosorbent assays (ELISAs) for detection of both the protective antigen (PA) and lethal factor (LF) components of the anthrax exotoxin in serum. The assays utilize as capture agents an engineered high-affinity antibody to PA, a soluble form of the extracellular domain of the anthrax toxin receptor (ANTXR2/CMG2), or PA itself. Sandwich immunoassays were used to detect and quantify PA and LF in animals infected with the Ames or Vollum strains of anthrax spores. PA and LF were detected before and after signs of toxemia were observed, with increasing levels reported in the late stages of the infection. These results represent the detection of free PA and LF by ELISA in the systemic circulation of two animal models exposed to either of the two fully virulent strains of anthrax. Simple anthrax toxin detection ELISAs could prove useful in the evaluation of potential therapies and possibly as a clinical diagnostic to complement other strategies for the rapid identification of B. anthracis infection.

scholarly journals Anthrax Toxin-Mediated Delivery In Vivo and In Vitro of a Cytotoxic T-Lymphocyte Epitope from Ovalbumin

1998 ◽  
Vol 66 (2) ◽  
pp. 615-619 ◽  
Author(s):  
Jimmy D. Ballard ◽  
Amy M. Doling ◽  
Kathryn Beauregard ◽  
R. John Collier ◽  
Michael N. Starnbach
Keyword(s):  
Fusion Protein ◽  
T Lymphocyte ◽  
Self Assembly ◽  
Protective Antigen ◽  
Cytotoxic T Lymphocyte ◽  
Ex Vivo ◽  
Lethal Factor ◽  
Anthrax Toxin

ABSTRACT We reported earlier that a nontoxic form of anthrax toxin was capable of delivering a cytotoxic T-lymphocyte (CTL) epitope in vivo, such that a specific CTL response was primed against the epitope. The epitope, of bacterial origin, was fused to an N-terminal fragment (LFn) from the lethal-factor component of the toxin, and the fusion protein was injected, together with the protective antigen (PA) component, into BALB/c mice. Here we report that PA plus LFn is capable of delivering a different epitope—OVA257–264 from ovalbumin. Delivery was accomplished in a different mouse haplotype,H-2Kb and occurred in vitro as well as in vivo. An OVA257–264-specific CTL clone, GA-4, recognized EL-4 cells treated in vitro with PA plus as little as 30 fmol of the LFn-OVA257–264 fusion protein. PA mutants attenuated in toxin self-assembly or translocation were inactive, implying that the role of PA in epitope delivery is the same as that in toxin action. Also, we showed that OVA257–264-specific CTL could be induced to proliferate by incubation with splenocytes treated with PA plus LFn-OVA257–264. These findings imply that PA-LFn may serve as a general delivery vehicle for CTL epitopes in vivo and as a safe, efficient tool for the ex vivo expansion of patient-derived CTL for use in adoptive immunotherapy.

scholarly journals Anthrax Spores Make an Essential Contribution to Vaccine Efficacy

2002 ◽  
Vol 70 (2) ◽  
pp. 661-664 ◽  
Author(s):  
Fabien Brossier ◽  
Martine Levy ◽  
Michèle Mock
Keyword(s):  
Guinea Pigs ◽  
Protective Antigen ◽  
Humoral Response ◽  
Anthrax Toxin ◽  
Experimental Animals ◽  
Challenge Strain ◽  
Anthrax Spores ◽  
Acellular Vaccines ◽  
Essential Contribution ◽  
Anthrax Vaccines

ABSTRACT Anthrax is caused by Bacillus anthracis, a gram-positive spore-forming bacterium. Septicemia and toxemia rapidly lead to death in infected mammal hosts. Currently used acellular vaccines against anthrax consist of protective antigen (PA), one of the anthrax toxin components. However, in experimental animals such vaccines are less protective than live attenuated strains. Here we demonstrate that the addition of formaldehyde-inactivated spores (FIS) of B. anthracis to PA elicits total protection against challenge with virulent B. anthracis strains in mice and guinea pigs. The toxin-neutralizing activities of sera from mice immunized with PA alone or PA plus FIS were similar, suggesting that the protection conferred by PA plus FIS was not only a consequence of the humoral response to PA. A PA-deficient challenge strain was constructed, and its virulence was due solely to its multiplication. Immunization with FIS alone was sufficient to protect mice partially, and guinea pigs totally, against infection with this strain. This suggests that spore antigens contribute to protection. Guinea pigs and mice had very different susceptibilities to infection with the nontoxigenic strain, highlighting the importance of verifying the pertinence of animal models for evaluating anthrax vaccines.

Deletion mutants of protective antigen that inhibit anthrax toxin both in vitro and in vivo

2003 ◽  
Vol 307 (3) ◽  
pp. 446-450 ◽  
Author(s):  
Nidhi Ahuja ◽  
Praveen Kumar ◽  
Sheeba Alam ◽  
Megha Gupta ◽  
Rakesh Bhatnagar
Keyword(s):  
Protective Antigen ◽  
Anthrax Toxin ◽  
Deletion Mutants

scholarly journals Vaccination against Anthrax with Attenuated Recombinant Strains of Bacillus anthracis That Produce Protective Antigen

1999 ◽  
Vol 67 (2) ◽  
pp. 562-567 ◽  
Author(s):  
John P. Barnard ◽  
Arthur M. Friedlander
Keyword(s):  
Bacillus Anthracis ◽  
Protective Antigen ◽  
Plasmid Stability ◽  
Protective Efficacy ◽  
Live Vaccines ◽  
Antibody Titers ◽  
Anthrax Protective Antigen ◽  
Anthrax Vaccines

ABSTRACT The protective efficacy of several live, recombinant anthrax vaccines given in a single-dose regimen was assessed with Hartley guinea pigs. These live vaccines were created by transforming ΔANR and ΔSterne, two nonencapsulated, nontoxinogenic strains of Bacillus anthracis, with four different recombinant plasmids that express the anthrax protective antigen (PA) protein to various degrees. This enabled us to assess the effect of the chromosomal background of the strain, as well as the amount of PA produced, on protective efficacy. There were no significant strain-related effects on PA production in vitro, plasmid stability in vivo, survival of the immunizing strain in the host, or protective efficacy of the immunizing infection. The protective efficacy of the live, recombinant anthrax vaccine strains correlated with the anti-PA antibody titers they elicited in vivo and the level of PA they produced in vitro.

scholarly journals Protection against Anthrax Toxemia by Hexa-d-Arginine In Vitro and In Vivo

2004 ◽  
Vol 72 (1) ◽  
pp. 602-605 ◽  
Author(s):  
Miroslav S. Sarac ◽  
Juan R. Peinado ◽  
Stephen H. Leppla ◽  
Iris Lindberg
Keyword(s):  
Therapeutic Intervention ◽  
Protective Antigen ◽  
Proteolytic Cleavage ◽  
Anthrax Toxin ◽  
In Cells

ABSTRACT The anthrax toxin protective antigen precursor is activated by proteolytic cleavage by furin or a furin-like protease. We present here data demonstrating that the small stable furin inhibitor hexa-d-arginine amide delays anthrax toxin-induced toxemia both in cells and in live animals, suggesting that furin inhibition may represent a reasonable avenue for therapeutic intervention in anthrax.

Identification of Therapeutically Active Molecules against Anthrax through Structure and Ligand based Drug Design

2019 ◽  
Vol 18 (27) ◽  
pp. 2294-2312
Author(s):  
Sisir Nandi ◽  
Mridula Saxena ◽  
Anil Kumar Saxena
Keyword(s):  
Drug Design ◽  
Protective Antigen ◽  
Structural Features ◽  
Anthrax Toxin ◽  
Biological Warfare ◽  
Discovery Research ◽  
Drug Discovery Research ◽  
Pathogenic Viruses

Background: People suffer from fatal diseases which are responsible for mortality. Potent devices and medicines are being developed to fight diseases caused by the microorganism for saving the lives of individuals. Highly pathogenic viruses and bacteria are being incorporated into biological warfare, which has become a major threat to mankind and causes the destruction of lives in a short span of time. Objective: The pathogen Bacillus anthracis, which is the causative of anthrax, is used in bioterrorism. Efforts are therefore being made to study the progress of biodefense drug discovery research in combating anthrax-based bioterrorism. Methods: This review describes the present status of the studies ontherapeutic measurement of anthrax toxin inhibitors towards inhibition of protective antigen, lethal and edema factors using chemometric and drug design tools to explore essential structural features for further design of active congeneric compounds. Results: The inhibitors estimated to show high activity through different models may be proposed for further synthesis and testing of biological activity in terms of anthrax toxin inhibition and cytotoxicity testing by in vitro and in vivo assays. Conclusion: Such an attempt is an insight of biodefense drug design against the dreadful threat to the nation due to anthrax-based terrorism and biological warfare.

scholarly journals Different mechanisms of two anti-anthrax protective antigen antibodies and function comparison between them

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Siping Xiong ◽  
Tingting Zhou ◽  
Feng Zheng ◽  
Xudong Liang ◽  
Yongping Cao ◽  
...  
Keyword(s):  
Western Blot ◽  
Protective Antigen ◽  
Anthrax Toxin ◽  
Antibody Treatment ◽  
Anthrax Protective Antigen ◽  
And Function ◽  
F344 Rats ◽  
New Strategies

Abstract Background Bacillus anthracis causes a highly lethal infectious disease primarily due to toxin-mediated injury. Antibiotics are no longer effective to treat the accumulation of anthrax toxin, thereby new strategies of antibody treatment are essential. Two anti- anthrax protective antigen (PA) antibodies, hmPA6 and PA21, have been reported by our lab previously. Methods The mechanisms of the two antibodies were elucidated by Electrophoresis, Competitive Enzyme-linked immune sorbent assay, Western blot analysis and immunoprecipitation test, and in vitro, in vivo (F344 rats) treatment test. The epitopes of the two antibodies were proved by Western blot and Enzyme-linked immune sorbent assay with different domains of PA. Results In this study, we compared affinity and neutralization of these two antibodies. PA21 was better in protecting cells and rats, whereas hmPA6 had higher affinity. Furthermore, the neutralization mechanisms of the two antibodies and their recognition domains of PA were studied. The results showed that hmPA6 recognized domain IV, thus PA could not bind to cell receptors. Conversely, PA21 recognized domain II, thereby limiting heptamer oligomerization of PA63 in cells. Conclusions Our studies elucidated the mechanisms and epitopes of hmPA6 and PA21. The present investigation can advance future use of the two antibodies in anthrax treatment or prophylaxis, and potentially as a combination treatment as the antibodies target different epitopes.

scholarly journals Recombinant Anthrax Toxin Receptor-Fc Fusion Proteins Produced in Plants Protect Rabbits against Inhalational Anthrax

2010 ◽  
Vol 55 (1) ◽  
pp. 132-139 ◽  
Author(s):  
Keith L. Wycoff ◽  
Archana Belle ◽  
Dorothée Deppe ◽  
Leah Schaefer ◽  
James M. Maclean ◽  
...  
Keyword(s):  
Protective Antigen ◽  
Tobacco Plant ◽  
Anthrax Toxin ◽  
Lethal Challenge ◽  
Edema Toxin ◽  
Toxin Receptor ◽  
Plant Expression Systems ◽  
Mutant Forms

ABSTRACTInhalational anthrax, a zoonotic disease caused by the inhalation ofBacillus anthracisspores, has a ∼50% fatality rate even when treated with antibiotics. Pathogenesis is dependent on the activity of two toxic noncovalent complexes: edema toxin (EdTx) and lethal toxin (LeTx). Protective antigen (PA), an essential component of both complexes, binds with high affinity to the major receptor mediating the lethality of anthrax toxinin vivo, capillary morphogenesis protein 2 (CMG2). Certain antibodies against PA have been shown to protect against anthraxin vivo. As an alternative to anti-PA antibodies, we produced a fusion of the extracellular domain of human CMG2 and human IgG Fc, using both transient and stable tobacco plant expression systems. Optimized expression led to the CMG2-Fc fusion protein being produced at high levels: 730 mg/kg fresh leaf weight inNicotiana benthamianaand 65 mg/kg inN. tabacum. CMG2-Fc, purified from tobacco plants, fully protected rabbits against a lethal challenge withB. anthracisspores at a dose of 2 mg/kg body weight administered at the time of challenge. Treatment with CMG2-Fc did not interfere with the development of the animals' own immunity to anthrax, as treated animals that survived an initial challenge also survived a rechallenge 30 days later. The glycosylation of the Fc (or lack thereof) had no significant effect on the protective potency of CMG2-Fc in rabbits or on its serum half-life, which was about 5 days. Significantly, CMG2-Fc effectively neutralized,in vitro, LeTx-containing mutant forms of PA that were not neutralized by anti-PA monoclonal antibodies.

scholarly journals Upstream sequence-dependent suppression and AtxA-dependent activation of protective antigens inBacillus anthracis

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6718 ◽  
Author(s):  
Kochi Toyomane ◽  
Yoshikazu Furuta ◽  
Daisuke Fujikura ◽  
Hideaki Higashi
Keyword(s):  
Molecular Mechanism ◽  
Protective Antigen ◽  
Dna Interaction ◽  
Anthrax Toxin ◽  
Upstream Region ◽  
Upstream Sequence ◽  
Mobility Shift ◽  
Vitro Analysis

The anthrax toxin is a virulence factor produced by the bacteriumBacillus anthracis. Transcription of anthrax toxin genes is controlled by the transcription factor AtxA. Thus, AtxA is thought to be a key factor for the pathogenicity ofB. anthracis. Despite its important role inB. anthracisinfection, the molecular mechanism by which AtxA controls expression of anthrax toxin remains unclear. This study aimed to characterize the molecular mechanism of AtxA-mediated regulation of protective antigen (PA), a component of anthrax toxin encoded by thepagAgene. First, the interaction between the upstream region ofpagAand AtxA was evaluated in vivo by constructing a transcriptional fusion of the upstream region with an auxotrophic marker. The results showed that (i) the upstream region ofpagAsuppressed transcription of the downstream gene and (ii) AtxA recovered suppressed transcription. Second, in vitro analysis using a gel mobility shift assay was performed to evaluate binding specificity of the AtxA–DNA interaction. The result showed sequence-independent binding of AtxA to DNA. Taken together, our findings suggest that the expression of PA was suppressed by the upstream region ofpagAand that an interaction of AtxA and the upstream region releases the suppression.

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